標題: 克雷白氏肺炎桿菌CG43中FimK、PecSM蛋白和CpxAR雙分子系統對第一型及第三型纖毛表現的調控角色探討
Characterization of the regulatory roles of FimK, PecSM, and CpxAR on the expression of type 1 and type 3 fimbriae in Klebsiella pneumoniae CG43
作者: 王哲充
Wang, Zhe-Chong
彭慧玲
Peng, Hwei-Ling
生物科技學系
關鍵字: 第一型纖毛;第三型纖毛;雙分子調控系統;克雷白氏肺炎桿菌;type 1 fimbriae;type 3 fimbriae;two component system;Klebsiella pneumoniae
公開日期: 2015
摘要: 克雷白氏肺炎桿菌是一株常在免疫力低下的病患身上造成化膿性潰瘍、菌血症、尿道及呼吸道感染的革蘭氏陰性伺機性病原菌。本論文以我們實驗室研究較多、源自林口長庚醫院肝膿瘍病患的克雷白氏肺炎桿菌CG43為探討菌株。 第一型纖毛和第三型纖毛是克雷白氏肺炎桿菌造成尿道感染或形成生物膜的重要因子,這兩套纖毛分別由fimAICDFGHK、fimB及fimE與mrkABCDF、mrkHI及mrkJ基因組轉譯出的蛋白組裝及調控表現。此二基因組fimAICDFGHK與mrkABCDF間隔約4.6 kb,而此DNA片段含有兩個基因分別可以轉譯為PecS轉錄因子及促進PecS活性的PecM穿膜蛋白,上述基因結構在已被發表的克雷白氏肺炎桿菌基因體都完整保留,本論文探討FimK、PecSM蛋白及CpxAR雙分子系統如何調控克雷白氏肺炎桿菌CG43第一型纖毛及第三型纖毛的表現。 在本論文的第2章,我們以及時定量聚合酶連鎖反應、啟動子活性分析以及電泳遷移率實驗,證實FimK能夠藉由蛋白N端區域與第一型纖毛啟動子DNA片段結合來促進第一型纖毛的表現量;此外,藉西方墨點法、細菌泳動能力測試與磷酸二酯酶活性分析,我們證實FimK蛋白C端片段具有分解環雙鳥糞嘌呤核甘酸(c-di-GMP)的能力;因此,我們推論FimK蛋白酵素活性的活化能夠降解細菌體內的c-di-GMP,進而間接抑制第三型纖毛的生合成。 第3章描述我們以西方墨點法、啟動子活性分析與電泳遷移率實驗證明CpxAR系統能夠直接負向調控PecS與PecM蛋白的表現,而PecS蛋白可能間接影響第一型纖毛的活性;此外,我們也證實尿酸鹽(urates)能夠調控PecS蛋白的活性;PecM的表現可降低尿酸鹽對克雷白氏肺炎桿菌CG43的生長抑制作用,我們推論PecM蛋白扮演傳輸尿酸鹽的角色,而藉降低細胞內的尿酸鹽含量來增進PecS的活性。 在第4章中,我們探討雙分子調控系統CpxAR如何影響第三型纖毛的表現。已有研究指出:克雷白氏肺炎桿菌細胞內c-di-GMP水平能夠影響第三型纖毛的表現,而磷酸二酯酶MrkJ及雙鳥苷酸環化酶YfiN能夠分別降解以及生合成c-di-GMP分子,進而調控第三型纖毛的生合成。因此,我們以及時定量聚合酶連鎖反應分析CpxAR基因缺損是否影響mrkJ或yfiN基因的轉錄水平。結果顯示yfiN基因的轉錄有顯著提升,此暗示CpxAR系統可能藉由負向調控影響雙鳥苷酸環化酶YfiN的表現,降低細胞內的c-di-GMP量而抑制第三型纖毛的表現;此外,CpxAR基因缺損提高克雷白氏肺炎桿菌對於氧化壓力及界面活性劑的敏感性,並改變外膜孔洞蛋白及纖維素生成的生成量;為了進一步探討Cpx系統是否經由改變外膜組成或是其他調控途徑而影響第三型纖毛的表現量,我們同時以核醣核酸定序技術來比較分析CG43S3與CG43S3cpxAR轉錄體,希望未來分析與驗證定序結果後,能讓我們更深入了解Cpx系統如何調控第三型纖毛的生成。
Klebsiella pneumoniae is a Gram-negative pathogen which causes suppurative lesions, bacteremia, and urinary as well as respiratory tract infections commonly in immunocompromised patients. K. pneumoniae CG43, which was isolated from a pyogenic liver abscess patient in Chang Gung hospital and has long been studied in our laboratory, was used in the investigation. Type 1 and type 3 fimbriae are crucial factors involved in K. pneumoniae urinary tract infection and biofilm formation. The encoding genes for biosynthesis and regulation are respectively fimAICDFGHK, and fimB and fimE for type 1 fimbriae, and mrkABCDF, mrkHI and mrkJ for type 3 fimbriae. The approximately 4.6 kb DNA, in-between fimAICDFGHK and mrkABCDF, contains homologous gene for a transcription regulator PecS and a transmembrane protein PecM for the PecS activity activation. The above-mentioned gene organization is conserved in all the published K. pneumoniae genomes. The thesis investigates how the regulatory proteins FimK, PecSM and the two component system CpxAR regulate the expression of type 1 and type 3 fimbriae in K. pneumonia CG43. In Chapter 2, we have proved using quantitative real time polymerase chain reaction (qRT-PCR), promoter activity assay and electrophoretic mobility shift assay (EMSA) that the N-terminal domain of FimK is able to enhance the type 1 fimbrial synthesis by directly binding to the fimA promoter. In addition, we have shown, as assessed using the western blot analysis, swimming and PDE activity measurement, that the C-terminal domain of FimK carries a phosphodiesterase (PDE) activity. Accordingly, we speculate that the activation of the FimK enzymatic activity may degrade the intracellular c-di-GMP thereafter inhibit the expression of type 3 fimbriae. Chapter 3 describes the use of western blot analysis, promoter activity measurement, and EMSA to demonstrate that CpxAR directly down-regulates the expression of PecS and PecM, and PecS probably represses the expression of type 1 fimbriae indirectly. In addition, we have shown that the regulatory activity of PecS could be attenuated by urates. Moreover, the expression of PecM could release the inhibitory effect of urates on the growth of K. pneumonia. This suggests a role of PecM in pumping urate out of the cells thereafter leading to PecS activation. In Chapter 4, we investigate how CpxAR regulatory system influences the expression of type 3 fmbriae. Prior studies have reported that the c-di-GMP level of K. pneumoniae influences the expression of type 3 fimbriation. The phosphodiesterase MrkJ and the diguanylate cyclase YfiN which are capable of degradation and synthesis of c-di-GMP, respectively, have been reported to be involved in regulating the type 3 fimbriation. We hence analyze using qRT-PCR to determine if the transcript level of mrkJ and yfiN is affected by the deletion of cpxAR. The result showed that the YfiN transcript level was significantly increased by cpxAR deletion suggesting CpxAR plays a negative role on the c-di-GMP level thereafter represses the expression of type 3 fimbriae. In addition, we have found that the deletion of cpxAR increases the sensitivity of K. pneumoniae to oxidative stress and detergents, alters the outer membrane porin profile and cellulose production. To investigate further if Cpx system influences the outer membrane profile or interacts with other regulatory systems to affect the type 3 fimbriae expression, we employ a comparative transcriptomic analysis of CG43S3 and CG43S3cpxAR by sequencing the RNA transcript. We expect the future analysis and clarification of the sequencing data would benefit us toward in depth understanding of the regulatory mechanism of CpxAR on the expression of type 3 fimbriae.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079728510
http://hdl.handle.net/11536/125844
Appears in Collections:Thesis